Development of New Leishmanicidal Compounds via Bioconjugation of Antimicrobial Peptides and Antileishmanial Guanidines.

Leishmaniasis refers to a collection of diseases caused by protozoa from the Leishmania genus. These diseases, along with other parasitic afflictions, pose a significant public health issue, particularly given the escalating number of at-risk patients. This group includes immunocompromised individuals and those residing in impoverished conditions. The treatment of leishmaniasis is crucial, particularly in light of the mortality rate associated with nontreatment, which stands at 20-30,000 deaths per year globally. However, the therapeutic options currently available are limited, often ineffective, and potentially toxic. Consequently, the pursuit of new therapeutic alternatives is warranted. This study aims to design, synthesize, and evaluate the leishmanicidal activity of antimicrobial peptides functionalized with guanidine compounds and identify those with enhanced potency and selectivity against the parasite. Accordingly, three bioconjugates were obtained by using the solid-phase peptide synthesis protocol. Each proved to be more potent against intracellular amastigotes than their respective peptide or guanidine compounds alone and demonstrated higher selectivity to the parasites than to the host cells. Thus, the conjugation strategy employed with these compounds effectively contributes to the development of new molecules with leishmanicidal activity.

New Strategies for Novel Drugs: Antimicrobial Peptides Containing Ferrocene with Improved Antifungal and Antiplasmodial Biological Activity.

BACKGROUND: Fungal and parasitic diseases are global health problems, and the available treatments are becoming ineffective, mainly due to the emergence of resistant strains of pathogens. Furthermore, the drugs currently in use exhibit high toxicity and side effects. The scarcity of efficient treatments for fungal and parasitic diseases has motivated the search for new drug candidates, including antimicrobial peptides. The chemokine class RP1 peptide shows inhibitory activity against bacteria, viruses, cancer cells and parasites. In addition, the organometallic compound ferrocene showed antiparasitic activity. OBJECTIVE: Study aimed to assess the effect of conjugation of the RP1 peptide with ferrocene in terms of its structure, biological activity against fungi and parasites and toxicity. METHODS: Peptides and conjugates were synthesized using solid phase peptide synthesis (SPPS). The Fc-RP1 peptide showed antifungal and antimalarial activities with low toxicity in the U87 and HepG2 cell lines. RESULTS: The mechanism of action of these peptides, analyzed by flow cytometry in the fungus Cryptococcus neoformans, was through membrane permeabilization, with an emphasis on the Fc-RP1 peptide that presented the highest rate of PI-positive cell marking. CONCLUSION: In conclusion, ferrocene conjugated to antimicrobial peptide RP1 is an attractive biomolecule for drug discovery against fungal and parasitic diseases.

Antimicrobial activity of RP-1 peptide conjugate with ferrocene group.

Parasitic diseases are a neglected and serious problem, especially in underdeveloped countries. Among the major parasitic diseases, Leishmaniasis figures as an urgent challenge due to its high incidence and severity. At the same time, the indiscriminate use of antibiotics by the population is increasing together with resistance to medicines. To address this problem, new antibiotic-like molecules that directly kill or inhibit the growth of microorganisms are necessary, where antimicrobial peptides (AMPs) can be of great help. In this work, the ferrocene molecule, one active compound with low levels of in vivo toxicity, was coupled to the N-terminus of the RP1 peptide (derived from the human chemokine CXCL4), aiming to evaluate how this change modifies the structure, biological activity, and toxicity of the peptide. The peptide and the conjugate were synthesized using the solid phase peptide synthesis (SPPS). Circular dichroism assays in PBS showed that the RP1 peptide and its conjugate had a typical spectrum for disordered structures. The Fc-RP1 presented anti-amastigote activity against Leishmania amazonensis (IC50 = 0.25 µmol L-1). In comparison with amphotericin B, a second-line drug approved for leishmaniasis treatment, (IC50 = 0.63 µmol L-1), Fc-RP1 was more active and showed a 2.5-fold higher selectivity index. The RP1 peptide presented a MIC of 4.3 µmol L-1 against S. agalactiae, whilst Fc-RP1 was four times more active (MIC = 0.96 µmol L-1), indicating that ferrocene improved the antimicrobial activity against Gram-positive bacteria. The Fc-RP1 peptide also decreased the minimum inhibitory concentration (MIC) in the assays against E. faecalis (MIC = 7.9 µmol L-1), E. coli (MIC = 3.9 µmol L-1) and S. aureus (MIC = 3.9 µmol L-1). The cytotoxicity of the compounds was tested against HaCaT cells, and no significant activity at the highest concentration tested (500 µg. mL-1) was observed, showing the high potential of this new compound as a possible new drug. The coupling of ferrocene also increased the vesicle permeabilization of the peptide, showing a direct relation between high peptide concentration and high carboxyfluorescein release, which indicates the action mechanism by pore formation on the vesicles. Several studies have shown that ferrocene destabilizes cell membranes through lipid peroxidation, leading to cell lysis. It is noteworthy that the Fc-RP1 peptide synthesized here is a prototype of a bioconjugation strategy, but it still is a compound with great biological activity against neglected and fish diseases.

Identification and characterization of an antimicrobial peptide of Hypsiboas semilineatus (Spix, 1824) (Amphibia, Hylidae).

The multidrug-resistant bacteria have become a serious problem to public health. In this scenery the antimicrobial peptides (AMPs) derived from animals and plants emerge as a novel therapeutic modality, substituting or in addition to the conventional antimicrobial. The anurans are one of the richest natural sources of AMPs. In this work several cycles of cDNA cloning of the skin of the Brazilian treefrog Hypsiboas semilineatus led to isolation of a precursor sequence that encodes a new AMP. The sequence comprises a 27 residue signal peptide, followed by an acidic intervening sequence that ends in the mature peptide at the carboxy terminal. The AMP, named Hs-1, has 20 amino acids residues, mostly arranged in an alpha helix and with a molecular weight of 2144.6 Da. The chemically synthesized Hs-1 showed an antimicrobial activity against all Gram-positive bacteria tested, with a range of 11-46 µM, but it did not show any effect against Gram-negative bacteria, which suggest that Hs-1 may have a selective action for Gram-positive bacteria. The effects of Hs-1 on bacterial cells were also demonstrated by transmission electron microscopy. Hs-1 is the first AMP to be described from H. semilineatus.